Lab 4: Allelic Typing of ABO Gene
LAB 4: ALLELIC TYPING OF THE ABO GENE BY PCR
I. General Introduction
On June 26, 2000 it was officially announced that the sequencing of the entire human
genome had been completed. Deciphering the 3 billion base pairs of information
provided by this project, and attempting to understand how this information is integrated
and used during the growth and development of the human organism, will keep scientists
busy for decades to come. But we do not have to wait for a full understanding of the
workings of the human genome to take advantage of the wealth of sequence information
that resulted from this project. This information and the powerful tools of molecular
biology are already providing us with novel and sophisticated methods for augmenting
medical practices. One example of this is the detection of disease causing alleles in
adults or unborn fetuses to give parents the information they need to make difficult
medical decisions and to help physicians better tailor medical procedures for the
individual. To illustrate how allelic typing can be accomplished, we will each determine
our allelic composition (genotype) for the gene that determines our ABO blood type.
This procedure relies on PCR (the polymerase chain reaction), a technique that has
revolutionized molecular biology and won Kary Mullis the 1993 Nobel Prize in
chemistry. With this technique it is possible to specifically amplify one gene out of the
entire genome, and produce enough copies of it to determine its nucleotide sequence by
standard biochemical procedures. In our exercise we will amplify the ABO gene from
total genomic DNA isolated from cheek epidermal cells. The amplification product will
be purified and a sample of it will be examined by agarose gel electrophoresis. Upon
verification that the amplification and purification were successful, it will be given to the
FSU Sequencing Facility where it will be sequenced using automated technology. A
comparison of this sequence with sequences published in the literature will enable you to
determine the allele(s) you possess for the ABO gene.
II. Day 1: DNA Isolation and PCR
A. Introduction
In today’s lab you will isolate DNA from a sample of your cheek cells and perform PCR
to amplify the ABO gene. By vigorously rinsing your mouth with an isotonic saline
solution, you should be able to collect several thousand cheek cells. Each of these cells
contains 6.5 pg of DNA, but only a small fraction of this DNA corresponds to the
segment of the ABO gene that we are investigating (less than 0.000007%). Even if you
collect 10,000 cheek cells, you are starting your PCR reactions with less than 0.001 pg of
DNA corresponding to the ABO gene. Nevertheless, after amplification you will have
approximately 1 g of pure ABO segment which is more than sufficient for
electrophoresis and DNA sequencing. The polymerase chain reaction is described in
Appendix 1, and the molecular biology of the ABO gene is described in Appendix 2.
Both appendices should be studied before starting this lab.
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Lab 4: Allelic Typing of ABO Gene
B. Materials
gloves
sterile 1.5 and 0.5 ml microfuge tubes
extra fine point Sharpies
clean Pipetman and tips
clean tube racks and ice buckets
clean microcentrifuge
boiling water bath
thermocycler
sterile pipettes and pipette fillers
sterile disposable drinking cups
ice and ice buckets
0.9% NaCl (physiological saline)
10% Chelex in H2O
sterile mineral oil
95% ethanol in squirt bottles
dilute Taq polymerase (2 U/10 l: 0.4 l Taq polymerase + 9.6 l H2O per reaction)
PCR reaction mix (for each reaction)
22.25 l H2O
5.0 l 10X Taq Polymerase buffer
1.25 l 50 mM MgCl2 (2 mM MgCl2 in final reaction)
0.5 l 20 mM dNTPs (200 M each in final reaction)
2.0 l primer 3517 at 100 ng/l (200 ng)
2.0 l primer 3518 at 100 ng/l (200 ng)
C. Procedure (wear gloves throughout this procedure)
1. Label two 1.5 ml microcentrifuge tubes with your initials, and a third with your
initials and “cheek DNA.” Also label a sterile drinking cup with your initials.
2. With a sterile pipette, transfer 10 ml of 0.9% NaCl into the drinking cup.
3. Pour the saline into your mouth and swish vigorously from cheek to cheek for 45 sec
to 1 min.
4. Expel the saline back into the same drinking cup.
5. With P1000 Pipetman, transfer 1 ml of your cheek cell solution into each of the two
1.5 ml microcentrifuge tubes labeled just with your initials. Make sure you briefly
swirl the solution in the drinking cup before taking your samples.
6. Spin the tubes for 1 min in the microcentrifuge.
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Lab 4: Allelic Typing of ABO Gene
7. Carefully remove the supernatants with a P1000 Pipetman set to 1 ml. Do not disturb
the pellets, and leave just enough fluid (~25 l) to completely cover the cell pellets.
Discard the supernatants into appropriate waste containers.
8. Resuspend one of the pellets in the remaining fluid by pipetting the fluid and pellet up
and down with a P200 Pipetman set to 25 l.
9. Transfer the fluid and resuspended pellet to the tube with your other cell pellet, and
resuspend the pellet in that tube. You should now have ~50 l of fluid in one tube
containing both of your resuspended pellets.
10. Vortex the 10% Chelex solution to get an even distribution of Chelex throughout the
suspension, and then immediately transfer 200 l of the Chelex suspension to your
resuspended pellet using a P1000 Pipetman.
11. Vortex your tube for a few seconds, poke a hole in its top as instructed by your TA,
and then incubate in a boiling water bath for 10 min. This breaks open the cheek cells
and releases the DNA into solution.
12. Carefully remove the tube from the boiling water bath (don’t burn yourself), vortex it
for a few seconds, and then spin it for 1 min in the microcentrifuge.
13. There should be a pellet at the bottom of your tube that consists of cellular debris and
the Chelex particles. (The Chelex particles bind metal ions and other cellular factors
that would inhibit the PCR reaction.) Avoid this pellet and transfer 100 l of the
supernatant to the tube labeled with your initials and “cheek DNA.”
14. Label a 0.5 ml microcentrifuge tube with your initials and the number 1 and another
with your initials and the number 2. Write your labels in two places on these tubes;
once on the sides but near the top, and once on the actual tops of the tubes.
15. Using a P200 Pipetman, transfer 33 l of the PCR reaction mix that has been prepared
by your instructor to each of your 0.5 ml tubes. (Your TA might do this for you to
avoid wasting this precious mixture.)
16. Using a P20 Pipetman, add 7 l of your cheek cell DNA preparation to each of your
tubes. Do not discard the tube with the remainder of your cheek cell DNA
preparation. You will need this later. Instead, store it at 4oC as directed by your TA.
17. Firmly hold the tubes at their top and flick the bottoms with your index finger to mix.
Spin for a few seconds in the microcentrifuge. (Make sure you use the 0.5 ml tube
adaptors that should already be in place in the microcentrifuge.) Overlay 40 l of
mineral oil to each tube by slowly pipetting the oil onto the inner wall of the
microcentrifuge tube just above the level of the reaction mixture. Spin for a few
seconds in the microcentrifuge. Keep the tubes at room temperature.
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Lab 4: Allelic Typing of ABO Gene
18. When all students have set up their reactions, the tubes will be put on the
thermocycler. The TA will include a control reaction tube with NO DNA. After the
initial heating at 94oC, the instructor will add 10 l (2 units) of dilute Taq polymerase
to each tube. The complete program for amplifying the ABO gene is as follows:
Hot Start
94oC 3 min (to denature the double-stranded DNA)
80oC 6 min (to keep the strands separated while adding the enzyme)
Thermocycle (x30)
94oC 30 sec (denature)
60oC
1 min (anneal primers)
72oC
1 min (extension)
Final extension
72oC
7 min
4oC
hold
19. Upon completion of the program, the TA will store your tubes at 4oC.
III. Day 2: Product Purification
A. Introduction
The ultimate goal of this lab is to sequence the 213 bp fragment that was amplified from
the ABO gene. The nucleotides and primers remaining in the PCR reaction would
interfere with the sequencing reactions, so the amplified product needs to be purified
from these unwanted substances. This will be accomplished using QIAquick columns
made specifically for PCR purification. The column material is silica glass which binds
DNA at high ionic strength. Diluting your sample in buffer PB raises the ionic strength to
a level that causes the amplification product to bind to the column. While the DNA is
bound to the column, it is washed with another buffer of high ionic strength (buffer PE)
to remove all traces of unwanted substances from the reaction. Finally, the column is
flushed with water which causes the DNA to become unbound and elute into a collection
tube. The amplification product is now pure and ready to sequence.
Because DNA sequencing is a relatively expensive procedure, we want to make sure you
have enough material to make an attempt at sequencing worthwhile. Next lab period, we
will get a rough estimate of the success of your amplifications by analyzing aliquots of
your reactions by gel electrophoresis. For this purpose you will prepare samples of your
PCR reactions before the QIAquick column purifications, and another of the purified
product, that will be used for electorphoresis next period.
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Lab 4: Allelic Typing of ABO Gene
B. Materials
Pipetman, tips, 1.5 ml microfuge tubes, racks
microcentrifuge and vortex
ice bucket and ice
gel juice
QIAquick columns and collection tubes
QIAquick buffers PB and PE
ddH2O
C. Procedure (also see flow chart at the end of this procedure)
1. Label six 1.5 ml microfuge tubes with your initials and the following:
PCR1 gel
PCR2 gel
PCR combined
ABO elutant (Use a capless 1.5 ml tube for this)
elutant gel
Cheek DNA, no PCR
2. Retrieve your PCR reaction tubes from the 4oC refrigerator and give them a pop spin.
Use the centrifuge labeled “after PCR” for this and all subsequent steps. Transfer 10
l of one of your PCR reactions to the PCR1 gel tube, and 10 l of your other PCR
reaction to the PCR2 gel tube. Take your samples from the lower layer (aqueous
layer) in the reaction tubes and try not to suck up any of the mineral oil (do not
release the plunger on the Pipetman until the tip is completely past the oil and into
the aqueous layer). Remove excess oil from the tip by scraping it against the inside
of the tube opening as you pull out of the tube.
3. Add 1.5 l of gel juice to the PCR1 gel and PCR2 gel tubes. Vortex briefly, spin for
5 seconds and store on ice. These are “before column” samples of your PCR
reactions to examine by gel electrophoresis.
4. Transfer 35 l from what remains in each of the PCR reaction tubes to the PCR
combined tube. Again, try to avoid transferring any mineral oil with your samples.
The 70 l in this tube represents the bulk of your PCR reactions and will be purified
by passage over a QIAquick column.
5. Add 5 volumes of Buffer PB to 1 volume of your combined PCR reaction. If you
were able to collect 70 l of combined reactions, you would add 350 l of Buffer
PB. Briefly vortex and spin for 5 sec.
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Lab 4: Allelic Typing of ABO Gene
6. Obtain a QIAquick column. Make sure that it comes attached to a 2-ml collection
tube. Label the column and the collection tube with your initials. Apply your PCR
reaction + PB to the center of the top of the column matrix with a P1000 Pipetman.
Centrifuge for 1 min.
7. Discard the fluid in the collection tube, and put the column back into the same tube.
8. Wash the DNA bound to the column by adding 0.75 ml Buffer PE to the column and
centrifuging for 1 min.
9. Discard the flow through, replace column into same collection tube, and centrifuge
for an additional 1 min. Discard the collection tube.
10. Place QIAquick column with bound DNA into the capless tube labeled ABO elutant.
Add 50 l ddH2O to the center of the column matrix and centrifuge for 1 min. The
flow through now contains your purified PCR product.
11. Transfer 10 l from the ABO elutant tube to the tube labeled elutant gel. Add 1.5 l
of gel juice to this sample for the gel, vortex, and centrifuge for 5 sec. Store on ice.
Insert a cap onto the ABO elutant tube, and store this tube as directed by the TAs.
12. Retrieve your original cheek DNA preparation from the refrigerator. Vortex and pop
spin this tube and then transfer 10 l of it to the tube labeled cheek DNA, no PCR.
Add 1.5 l of gel juice, vortex, and pop spin. Store on ice.
13. You should now have four tubes of samples on ice that will be examined by
electrophoresis next period. Make sure these tubes are properly labeled, and store
them as directed at 4oC.
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Lab 4: Allelic Typing of ABO Gene
QIAquick Flow Chart
35 l
5 volumes
Buffer PB
35 l
load all onto column
QIAquick column
matrix = silica glass
binds DNA at high ionic
strength (high salt)
50 l
PCR
reactions
10 l +
1.5 l
gel juice
PCR combined
Centrifuge 1 min
DNA bound to column
flow through - discard
add 0.75 ml Buffer PE
Centrifuge 1 min (washes column)
PCR1 and PCR2
(save for gel)
DNA still bound to column
flow through - discard
Centrifuge 1 min
remove residual fluid
Put column into 1.5 ml tube
labeled ABO elutant
10 l + 1.5 l gel juice
Add 50 l ddH20
centrifuge 1 min
DNA in H20 - ABO elutant
give to TAs after taking
sample for gel
elutant gel
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Lab 4: Allelic Typing of ABO Gene
IV. Day 3: Electrophoresis
A. Introduction
Today you will examine the samples you prepared last lab period by electrophoresis on a
2.5% agarose gel. The results will help us determine how much of your purified product
to use for the sequencing reactions.
B. Materials
Pipetman, tips, tubes, and racks
microcentrifuge and vortex
materials and equipment for 2.5% agarose gel electrophoresis (see Day 3 of Lab 1)
C. Procedure
You should have four tubes of samples that you prepared last lab period to examine by
electrophoresis. A 2.5% agarose gel in 1x TAE will be prepared for this purpose. Load
10 l of your samples into the lanes assigned to you by the TAs. A photograph of the
finished gel will be given to you during the next lab meeting. From these results we will
determine how much of your ABO elutant is needed for DNA sequencing.
V. Sequencing
You will be instructed how much of your ABO elutant to put into a clean 1.5 ml
microfuge tube to give to the DNA Sequencing Facility. The staff at this facility will
perform automated sequencing of your amplification product using methods described in
Appendix 3.
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